Transcript Profiles of Leaf Development Indicate Co-ordinated Cell Development and Maturation
To obtain a broader understanding of developmental processes in wild type plants we conducted a transcriptomic comparison of the base, middle and tip regions of seven day old leaves. One-way analysis of variance identified 440 transcripts that were significantly differentially abundant (P< 0.05) in the different leaf regions. Transcripts were subjected to hierarchical clustering analysis which revealed five major clusters (Fig. 3). Cluster A comprised 69 transcripts that exhibited a gradient of abundance from low in the leaf base to high in the tip. This cluster included six transcripts encoding transcription factors homologous to Arabidopsis transcripts that have been shown to have roles in leaf development (Table S1). MLOC_74058.1 exhibits homology to an Arabidopsis transcription factor NGATHA3 (AT1G01030) involved in the control of leaf shape and expressed in leaf tips under the control of TCP (TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTOR) transcription factors (Ballester et al., 2015). The latter family were represented by MLOC_14785.1 which exhibited homology to Arabidopsis TCP5 (AT5G60970). A gene (MLOC_70809.1) encoding a homologue of Arabidopsis GATA, NITRATE-INDUCIBLE, CARBON METABOLISM INVOLVED (GNC) transcription factor (AT5G56860) that regulates stomatal development, greening and chloroplast development (Bastakis, Hedtke, Klermund, Grimm, & Schwechheimer, 2018; Klermund et al., 2016; Zubo et al., 2018) was also present in cluster A.
Two of the transcription factors identified within cluster A (Fig. 3) were associated with the control of senescence in response to metabolic signals. AK373121 exhibits homology to an Arabidopsis zinc finger family protein METHYLENE BLUE SENSITIVITY 1 (MBS1; AT3G02790) responsible for acclimation or cell death in dose-dependent response to1O2 (Shumbe et al., 2017) while MLOC_64240.2 and MLOC_53744.1 both share homology to AT1G56010 encoding NAC1, a senescence associated transcription factor under the control of auxin (Kim et al., 2011). Further evidence for the upregulation of senescence-associated processes in the leaf tip was the increased abundance of transcripts (AK370424, MLOC_47161.1) encoding proteins with homology to AUXIN-INDUCED IN ROOT CULTURES 3 (AT2G04160) and SENESCENCE-ASSOCIATED GENE 12 (AT5G45890; SAG12), endopeptidases required for protein turnover (James et al., 2018; Neuteboom, Veth-Tello, Cludesdale, Hooykaas, & van der Zaal, 1999). Furthermore, several transcripts (MLOC_56129.2, MLOC_57630.1, AK374126) encoding proteins homologous to proteins required for ubiquitin mediated protein turnover exhibited greatest abundance in the leaf tip (Table S1).
Cluster B was the largest of the clusters comprising 187 transcripts that exhibited a gradient of abundance from high to low from the leaf base to the leaf tip (Fig. 3). Seventeen transcripts encoding transcription factors were identified, several of which exhibited homology to Arabidopsis transcripts with functions in photomorphogenesis and development. Two transcripts (AK364144, MLOC_73144.4) showed homology to Arabidopsis auxin response factors (AT4G30080, AT1G19220; ARF) with functions in leaf morphogenesis and development (Liu, Jia, Wang, & He, 2011; Schuetz, Fidanza, & Mattsson, 2019). Similarly, AK376150 and AK365841 are homologues of Arabidopsis genes encoding INDETRMINATE DOMAIN 15 (AT2G01940) and GATA TRANSCRIPTION FACTOR 2 (AT2G45050), with functions in leaf morphogenesis and photomorphogenesis, respectively (Cui et al., 2013; Luo et al., 2010). As described below, a feature of cluster B were large numbers of transcripts associated with lipid and wax metabolism. Interestingly, we identified a transcript (AK364135) with homology to an Arabidopsis transcript encoding the class I TCP transcription factor TCP14 (AT3G47620). In Arabidopsis class I TCP transcription factors including TCP14 are master regulators of cuticle biosynthesis (Camoirano et al., 2020) and are required for the induction of genes involved in gibberellin biosynthesis and cell expansion in response to temperature (Ferrero, Viola, Ariel, & Gonzalez, 2019). Similarly, several transcripts in cluster B were associated with polyphenol metabolism and a transcription factor (AK361986) homologous to Arabidopsis MYB4 (AT4G38620) which functions in the control of flavonoid biosynthesis (Wang et al., 2020) was also identified in this cluster.
Consistent with the hypothesis that cells at the leaf base were undergoing division and expansion, 14 transcripts categorised as cell wall associated were identified in cluster B (Table S1). These included several transcripts (AK248822.1, AK356936, MLOC_36439.1, MLOC_43237.1, MLOC_12096.1, MLOC_73204.3) with homology to transcripts encoding Arabidopsis expansins with a well-established role in cell wall loosening, leaf initiation and subsequent growth (Marowa, Ding, & Kong, 2016). A further three transcripts (MLOC_61972.1, AK361522, AK361278) encoded xyloglucan endotransglycosylases (XTHs) that function in cell expansion by loosening cell walls (Rose, Braam, Fry, & Nishitani, 2002). Furthermore, transcripts encoding two pectin modifying enzymes, a methylesterase (MLOC_54267.1) and an acetylesterase (MLOC_55102.5) were highly abundant in the leaf base.
Transcripts associated with lipid metabolism were also highly represented within cluster B, consistent with the hypothesis that active cuticle biosynthesis is occurring in the basal portion of the leaf. For example, MLOC_67622.1 and MLOC_45058.1 both exhibited homology to Arabidopsis transcripts encoding 3-KETOACYL-COA SYNTHASE 6 (KCS6, AT1G68530). Plants carrying mutations in KCS6 exhibited significant reductions in branched and unbranched long chain alkanes and alcohols in cuticular wax (Buster, & Jetter, 2017). Similarly, AK252678.1 shared homology with AT5G43760 encoding KCS20 a very long chain fatty acid synthase required for cuticular wax and root suberin biosynthesis (Lee et al., 2009). Two transcripts (MLOC_54056.1 and AK370579) shared homology with AT1G02205 encoding ECERIFERUM 1, mutants of which exhibit similar defects to KCS6 mutant lines (Buster, & Jetter, 2017). Taken together these data suggest that the basal portion of the leaf comprises actively dividing/expanding cells.
Cluster C (Fig. 3) comprised 43 transcripts that were abundant at the leaf base, scarce in the middle section of the leaf with intermediate abundance in the leaf tip. Cluster D comprised 127 transcripts that exhibited a similar high to low abundance profile during leaf maturation as observed for cluster B. However, the expression gradient in cluster D was considerably greater than observed for cluster B. Like cluster B, cluster D contained several transcripts encoding proteins associated with cell wall metabolism including expansins, XTHs and pectin modifying enzymes (Table S1). Cluster D additionally contained transcripts encoding proteins required for cellulose biosynthesis where two transcripts (MLOC_66568.3, MLOC_68431.4) exhibited homology to Arabidopsis cellulose synthases (AT5G44030, AT5G17420) and a further two transcripts (MLOC_7722.1, AK370617) exhibited homology to an Arabidopsis transcript encoding the membrane anchored COBRA-LIKE 4 (AT5G15630) which plays a key function in cellulose deposition (Brown, Zeef, Ellis, Goodacre, & Turner, 2005).
Furthermore, cluster D contained transcripts associated cell expansion, cell polarity, organ patterning and development. MLOC_53132.1 exhibited significant homology to Arabidopsis transcripts (AT4G08950) encoding EXORDIUM, a brassinosteroid responsive gene that acts upstream of wall-associated kinases and expansins to promote cell expansion (Schröder, Lisso, Lange, & Müssig, 2009). Indeed, a transcript (AK364262) with homology to the Arabidopsis transcript encoding WALL-ASSOCIATED KINASE 2 (AT1G21270) required for turgor driven cell expansion was also identified (Kohorn et al., 2006). Several transcripts associated with vascular development and patterning were present. MLOC_58644.1 and AK359559 exhibited homology to Arabidopsis AT2G34710 and AT5G62880 encoding PHABULOSA and ROP11, respectively which play roles in xylem patterning during early cellular differentiation (Müller et al., 2016; Nagashima et al., 2018). Furthermore, MLOC_69397.2 homologous to an Arabidopsis transcript (AT1G79430) encoding ALTERED PHLOEM DEVLOPMENT with a role promoting phloem development was present (Bonke, Thitamadee, Mähönen, hauser, & Helariutta, 2003). Finally, a transcript encoding a basic-helix-loop-helix transcription factor (MLOC_55768.1) with similarity to an Arabidopsis transcript encoding bHLH93 (AT5G65640) was also present. This transcription factor interacts with FAMA which controls differentiation of guard cells in the leaf epidermis (Ohashi-Ito and Bergmann, 2006). Taken together these results imply active cellular expansion and differentiation in the base of the leaf and indicate that these processes are complete in the more mature leaf regions. Transcripts in cluster E displayed a similar pattern of abundance to those in cluster C with minimum abundance in the mid region of the leaf.